Background The diagnosis of Human African Trypanosomiasis (HAT) typically involves two steps: a serological screen, followed by the detection of living trypanosome parasites in the blood or lymph node aspirate. Live parasites can, however, remain undetected in some seropositive individuals, who we hypothesize are infected with Trypanosoma brucei gambiense parasites in their extravascular dermis. Methods and findings To test this hypothesis, we conducted a prospective observational cohort study in the gambiense HAT (gHAT) focus of Forecariah, in the Republic of Guinea. 5,417 subjects in this disease foci underwent serological screening for gHAT. Of these individuals, 66 were enrolled into our study, of whom 40 were seronegative, 8 were seropositive but unconfirmed, and 18 confirmed gHAT cases. Enrolled individuals underwent a dermatological examination, and had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immuno-histological methods. In confirmed cases, dermatological symptoms were significantly more frequent, relative to seronegative controls. T. b. gambiense parasites were present in the blood of all confirmed cases but not in unconfirmed seropositive individuals. However, trypanosomes were detected in the dermis of all unconfirmed seropositive individuals and confirmed cases. After 6 and 20 months of treatment, dermal trypanosome numbers in skin biopsies of confirmed cases progressively reduced. Conclusions Our results thus highlight the skin as a potential reservoir for trypanosomes, with implications for our understanding of this disease's epidemiology in the context of its planned elimination and highlighting the skin as a novel target for gHAT diagnostics.